Cosmetic Medicine in Japan -東京大学美容外科- トレチノイン(レチノイン酸)療法、アンチエイジング(若返り)
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Characterization of wound drainage fluids as a source of soluble factors associated with wound healing: Comparison to platelet-rich plasma and potential use in cell culture


One of the requirements for successful cell-based therapy is the delivery of stem cells to target tissues after manipulations such as the expansion stem cell cultures or commitment of stem cells to a specific lineage. However, due to safety considerations such as transmission of viral or prion-related disease, the use of animal-derived serum, tissue extracts, enzymes and cytokines in these manipulations is undesirable, so autologous, human-derived substitutes for these animal products could be very useful. Several studies have examined the use of alternatives: Attempts were made to use human autoserum as a replacement for fetal bovine serum (FBS) (McAlinden et al., 2000), though its volume available is limited. Patient-derived fibrin glue (thrombin and fibrinogen) and platelet-rich plasma have also been used for cell culture or in clinical trials for enhancing tissue regeneration (Thor et al., 2005). Other studies have shown that growth factors derived from platelets can be used to stimulate cell proliferation (Ross et al., 1974; Lucarelli et al., 2003; Eppley et al., 2004), but platelets can not provide some other major growth factors such as b-FGF, KGF, and HGF (Sanchez et al, 2003). In this study, we examined the biochemical profiles of wound drainage fluids and compared them to those of serum derived from platelet-rich and platelet-poor plasma in order to assess whether these drainage fluids could be used as a supplement and/or replacement for animal-derived factors in situations in which cell culture would ideally be carried out in the absence of such animal-derived factors.
The growth factor b-FGF, which is an important endogenous stimulator of angiogenesis (Montesano et al., 1986) and cell proliferation (Nissen et al., 1996), is released from surrounding wounded tissues during an early phase of wound healing (Cordon-Cardo et al., 1990; Schulze-Osthoff et al., 1990). Cellular b-FGF is released by the lysis of epidermal cells (Takamiya et al., 2002), fibroblasts (Takamiya et al., 2002), and endothelial cells (Muthukrishnan et al., 1991) around the wound, and b-FGF bound up in the extracellular matrix is released by the action of various wound proteases (Bashkin et al., 1989; Ishai-Michaeli et al, 1990). KGF is expressed in the dermis during wound healing (Werner et al., 1992), and stimulates wound reepithelialization (Werner et al., 1994). HGF was independently discovered as a powerful mitogen for hepatocytes and as a stimulator of dissociation of epithelial cells (Werner et al., 2003). HGF-producing cells are found among those of mesenchymal origin, and HGF stimulates cell proliferation, cell migration (Matsumoto et al., 1991), and the production of matrix metalloproteinase (MMP) (Dunsmore et al., 1996) in keratinocytes.
Wound healing consists of several stages, including inflammation, new tissue formation, and tissue remodeling, eventually leading to at least partial regeneration of the wounded area (Werner et al., 2003). Various growth factors directly or indirectly control these wound-healing phenomena, and can be detected in cutaneous wound fluids (Grayson et al., 1993; Ono et al, 1995; Vogt et al., 1998), or in wound fluids obtained through surgical suction drains (Matsuoka et al., 1989; Baker et al., 2003; Karayiannakis et al., 2003). The fluid composition and the concentration of growth factors in the wound fluids change as healing progresses, and thus the fluids reflects the sequential phenomena that occur during wound healing. However, there is currently only limited information pertaining to the characterization of surgical drainage fluid with regard to growth factors and other soluble factors. Although a few previous reports have suggested that wound fluids have mitogenic and chemotactic effects (Chen et al., 1992; Nissen et al., 1996; Trengove et al. 2000), there has been limited information beyond that. Also, although it may be clinically feasible to use wound fluids for cell-based regenerative therapies, there is limited information on this point, and protocols for use of wound fluids have yet to be optimized.
The present study focuses on the characterization of subcutaneous wound fluids obtained through surgical suction drains. Such fluids are can be aseptically harvested with minimal morbidity: for example, adipose-derived stem cells can be isolated from liposuction aspirates and subcutaneous wound fluids can be simultaneously obtained by leaving a suction drainage tube in the subcutaneous cavity in the same surgery. We sequentially collected surgical drainage fluids from the subcutaneous space after plastic surgery, and characterized the fluids by examining wound healing-associated soluble factors such as electrolytes, cytokines, chemokines and matrix metalloproteinases (MMPs). We compared these characterization profiles with those of platelet-poor and platelet-rich plasma, which can be also easily obtained from patients, and all three types of fluids were assessed for their potential utility in cell culture.


Collection and preparation of suction drainage fluid samples
We collected drainage fluids and punctured fluids from subcutaneous wounds from 21 patients, who underwent liposuction and abdominoplasty (7 patients), liposuction (5 patients), breast augmentation (3 patients), foreign body excision in breasts (2 patients), reduction mammoplasty (1 patient), cartilage transplantation for microtia (1 patient), facelift (1 patient) and parotid gland excision (1 patient) at the University of Tokyo Hospital. Before samples were obtained, all patients gave signed informed consent, as approved by the ethical committee of University of Tokyo School of Medicine. Suction drains (J-vac drainage system, Johnson & Johnson, Cornelia, GA, USA) were subcutaneously inserted into operative wounds during surgery, after which wound fluid was continuously suctioned at 40-60 mmHg and aseptically collected in the storage bag of the suction system. The wound fluids were centrifuged at 2,000 g for 30 minutes, and supernatant fluids were frozen at -80°C. The frozen samples were thawed at 37°C before analysis. Fifty-two drainage fluid samples were obtained from 18 patients whose wounds exhibited normal healing (NH group). Nineteen samples harvested on day 0 or day 1 from 15 patients were referred to as Drainage Fluid-Early (DF-E), while 9 samples harvested on day 5 or day 6 from seven patients were referred to as Drainage Fluid-Late (DF-L). In addition, punctured fluid samples were obtained from 3 patients who underwent abdominoplasty and had subcutaneous seroma formation (SF group). These samples were harvested by puncturing the subcutaneous seroma on day 14 or later, and were included in our analyses as Punctured fluids (PF).
Collection and preparation of human sera from platelet-rich plasma and platelet-poor plasma.
Human platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from four healthy volunteers. Blood was drawn into two 200 ml blood bags containing 0.327% citric acid, 2.63% sodium citrate, 0.0275% adenine, 0.251% sodium dihydrogenphosphate and 2.9% D-glucose solution (blood bag CPD-adenineR, Terumo, Tokyo, Japan). To isolate PRP, bags were centrifuged at 200 g for 10 min in a large-capacity refrigerated centrifuge (KUBOTA 9810, KUBOTA Co., Tokyo, Japan), and to isolate PPP the bags were centrifuged at 5000 g for 5 min; in both instances the supernatant was harvested. PPP was harvested also from 11 of the aforementioned NH group patients. To obtain serum from PRP (designated SPRP) and from PPP (designated SPPP), 100 ml of PRP or PPP was drawn into a flask and 200 U of thrombin was added. The flask was agitated for 60 min at 37°C and then incubated overnight at 4 °C, after which the liquid component was drawn into 50 ml tube, centrifuged at 2000 g for 10 min, and supernatants were obtained as for SPRP and SPPP. The serum samples were frozen at -80°C and thawed at 37°C before analysis.
Biochemical analysis of serum and drainage fluid constituents
Preoperative serums and sequential drainage fluids obtained from three patients either before surgery or on day 0 to day 6 post-operation were subjected to biochemical analyses for total protein, albumin, sodium, potassium, chloride, calcium and iron. Analysis was performed by SRL, Inc. (Tachikawa, Japan), a commercial analysis service.
Quantitative assays for cytokines, chemokines, and matrix metalloproteinases associated with wound healing
Concentrations of various cytokine growth factors (PDGF-BB, EGF, TGF-β1, b-FGF, VEGF, HGF, KGF and IGF-1) and chemokines (IL-6 and IL-8) in drainage fluid samples, punctured fluids, PRP, and PPP were assayed using anti-human ELISA kits (QuantikineR, R&D Systems Inc., Minneapolis, MN), according to the manufacturer’s instructions. Levels of immunoreactive cytokines as reported by the ELISA assay were measured at 450 nm by a microplate reader (Model 550, Biorad Laboratories, Hercules, CA), and a standard curve was generated to determine growth factor concentrations (pg/mL). Levels of three matrix metalloproteinases (MMP-1, MMMP-8 and MMP-13) in drainage fluids were also measured using anti-human ELISA kits (Biotrak ELISA System, Amersham Biosciences, Piscataway, NJ).
Primary cell culture
Adipose-derived stromal cells (ASCs) were isolated from human lipoaspirates and cultured as previously described (Yoshimura et al., 2006). Briefly, the suctioned fat was digested with 0.075% collagenase in PBS for 30 min with agitation at 37oC. Mature adipocytes and connective tissues were separated from pellets by centrifugation (800 g, 10 min). Cell pellets were resuspended in erythrocyte lysis buffer (155mM NH4Cl, 10mM KHCO3, 0.1mM EDTA), incubated for 5 min at room temperature, resuspended again and passed through a 100-μm mesh filter (Millipore, MA, USA), and then plated at a density of 5 x 106 nucleated cells/100 mm plastic dish. Cells were cultured in M-199 medium containing 10% FBS at 37oC under 5% CO2 in a humidified incubator.
Human dermal fibroblasts were isolated from normal skin samples obtained from plastic surgery. The skin samples were cut into pieces of approximately 3 × 3 mm and treated with 0.25 % trypsin in PBS for 24 hour at 4°C. After removal of the epidermis, the connective tissue fragments were attached to 100 mm plastic dishes and cultured with DMEM containing 10% FBS. Primary fibroblasts appeared 4 to 7 days after the initiation of outgrowth cultures and became confluent after 2 to 3 weeks.
Cell proliferation assay using culture medium containing drainage fluids
Culture media (M199 for ASCs and DMEM for dermal fibroblasts) containing FBS and/or drainage fluids (DF-E [early] or DF-L [late]) was prepared at various concentrations. The drainage fluid samples were sterilized using a 0.22 μm filter (MILLEX GV Filter Unit, Millipore) before use. 5x104 cells were plated in 60mm dishes containing the prepared medium, and the medium was changed on the third and fifth days. The cell numbers were counted on the seventh day using a NucleoCounter (Chemometec, Allerod, Denmark), and average numbers were calculated from three different cultures of the cell types for each condition. The averages were normalized by calculating a ratio of cell numbers grown in each condition to cell numbers grown in the standard culture conditions (5% FBS without drainage fluid).
The Mann-Whitney U-test with the Bonferroni correction was used to compare concentrations of cytokines, chemokines and MMP among drainages fluids (DF-E and DF-L), punctured fluids, and SPPP. The Mann-Whitney U-test alone was used for comparison between SPPP and SPRP and also to compare cell numbers in various culture conditions. P < 0.05 was considered to be significant.


Biochemical profiles of drainage fluids and blood serum
We measured daily changes of electrolytes, albumin, and total protein in drainage fluids taken from three patients in the normal healing (NH) group from day 0 to day 6, and compared these values with the preoperative serum measurements of these factors from the same patients (Fig. 1). Concentrations of total protein and albumin in drainage fluids on day 0 were about 50% of those in preoperative serum, and both total protein and albumin gradually decreased to about 30% of that found in serum by day 6 (Fig. 1). Concentrations of Na+, K+, Cl-, Ca++, and Fe++ in drainage fluids were similar to those in serum and did not significantly change with time. The concentration of Ca++ in drainage fluids was about 60-70% of that in serum and changed very little from day 0 to day 6. The concentration of Fe++ in drainage fluid was extremely variable among different patients due to variations in an individual’s hemorrhage volume, but in general was substantially greater than that in serum, and tended to decrease slightly with time.
Cytokine concentrations in drainage fluids, SPRP and SPPP
Daily sequential changes in various cytokine growth factors in drainage fluids from six (VEGF, KGF) or seven (all other cytokines) patients are shown in Fig. 2, and data from early and late drainage fluids (DF-E, DF-L), puncture fluid (PF), serum from platelet-poor plasma (SPPP) and serum from platelet-rich plasma (SPRP) are summarized in Fig. 3. DF-E and DF-L were harvested from patients in the NH group, while PF was harvested from patients in the seroma formation (SF) group. Concentrations of b-FGF, PDGF, EGF and TGF-β were much higher in DF-E than in DF-L or SPPP. The concentrations in DF-E (mean± S.E.) and their relative abundance in DF-E vs. DF-L were as follows: b-FGF, 678.8 ± 100.6 pg/mL, 34.8x DF-L; EGF, 125.0 ± 18.6 pg/mL, 58.7x DF-L; PDGF, 267.7 ± 64.2 pg/mL, 72.7x DF-L; and TGF-β, 45.1 ± 8.4 pg/mL, 9.4x DF-L. In SPRP, B-FGF was present only at very low levels, while PDGF, EGF, and TGF-β were abundant, and were significantly higher in SPRP than in SPPP. Both DF-E and PF contained high levels of TGF-β, although the concentration of TGF-β in PF was on average lower than in SPRP. Concentrations of b-FGF, EGF and PDGF were sufficiently low in PF that they could not be detected.
In DF-L and in PF, concentrations of VEGF and HGF were higher than in DF-E and gradually increased with successive postoperative days, although KGF concentrations peaked at day 3 and then began to decrease, in contrast to VEGF and HGF concentrations, which steadily increased through day 14. PF contained significantly higher amounts of VEGF, HGF, and KGF than DF-E, and SPPP and SPRP contained much lower concentrations of VEGF, HGF, and KGF compared to DF-E, DF-L, and PF (Fig.3). IGF-1 did not change significantly with postoperative time, and the concentration of IGF-1 in DF-E and DF-L was significantly lower (by approximately 50%) compared to SPPP and SPRP.
Chemokine and MMP concentrations in drainage fluids, SPRP and SPPP
Daily sequential changes in the IL-6 and IL-8 chemokines and in MMPs (collagenases) were tracked in drainage fluids from either five (IL-6, IL-8) or four (MMPs) patients in the NH group. The summarized data for levels of these factors in DF-E, DF-L, PF, and SPRP are shown in Fig. 4 and Fig. 5. IL-6 was present at high concentrations in DF-E and decreased gradually in successive postoperative days, while IL-8 increased gradually. DF-E contained twice as much IL-6 as was found in DF-L, while DF-L contained twice as much IL-8 as was found in DF-E. Both IL-6 and IL-8 were detected in small amounts in SPRP, but not in SPPP, and PF contained both chemokines, although there were significant differences in PF chemokine levels vs. chemokine levels in either DF-E or DF-L (Fig. 4). In drainage fluids, levels of MMP-13 were low and did not change with time. The concentration of MMP-1 was also low and though it increased somewhat with time, it remained relatively slight. In contrast, MMP-8 increased in the early phase of wound healing, peaked on day 2-3, and then gradually decreased with time. In drainage fluids, MMP-8 was present in much higher amounts as compared to MMP-1 and MMP-13 (Fig. 5).
Cell proliferation assays using culture medium including the drainage fluids
In medium that had not been supplemented with drainage fluids, ASCs proliferated in a dose-dependent manner with regard to the concentration of FBS; the dose-dependent relationship was valid up to 10% FBS (Fig. 6A). When ASCs were grown in media containing 5% FBS, cell proliferation was significantly enhanced by the addition of DF-E at concentrations of ?1% or by the addition of DF-L at concentrations of ?2%. The increase in proliferation was dose-dependent with respect to the concentration of DF-E or DF-L: When 5% FBS and 5% DF-E were added to the medium, cell count increased to over five times that of the control (5% FBS alone), and was twice as high as the cell count for media containing 10% FBS alone. In medium lacking FBS, the addition of drainage fluids significantly enhanced ASC proliferation but were less effective compared to the same concentrations of FBS. We also examined dermal fibroblasts, which proliferated in a dose-dependent manner with respect to FBS up to concentrations of 10% FBS (Fig. 6B). The proliferation of dermal fibroblasts was enhanced by drainage fluids in the absence of FBS, though the enhancement of proliferation by the addition of drainage fluids to 5% FBS was not so striking compared to that of the ASCs. Table 1 shows summary data for the concentrations of growth factors, cytokines, and MMPs in DF-E, DF-L, SPPP, and SPRP. We suggest that this data could be used as a guide in choosing the appropriate fluid supplement for cell culture, based on the specific needs of a given cell line.
Comparison to previous studies
We compared our data on drainage fluids from subcutaneous wounds with data from previous studies on fluids from peritoneal drainages (Baker et al., 2003) and on wound fluids from split-thickness skin donor sites (Vogt et al., 1998) (Fig. 7). We found that the source of the wound fluid affected cytokine growth factor profiles: In general, skin wound fluids contained substantially lower levels of growth factors (bFGF, EGF, and TGF-β) compared to subcutaneous or peritoneal drainage fluids. Peritoneal drainage fluids contained over 100 times more TGF-β compared to subcutaneous and skin wound drainage fluids. Subcutaneous and peritoneal drainage fluids similarly contained higher concentrations of b-FGF, EGF, and VEGF. The difference in the cytokine profiles may reflect the requirement for different biochemical conditions for the optimal healing of each wound type.

Biochemical analysis of drainage fluids
Results from the analysis of the biochemical composition of drainage fluids in this study were similar to those of a previous study analyzing biochemical profiles of drainage fluids after axillary dissection (Bonnema et al., 1999). Our analysis showed that concentrations of Na+, K+, and Cl- in drainage fluids were similar to those in plasma, while concentrations of Ca++, total protein and albumin in drainage fluids were ~60-80% lower than those in plasma. The concentration of Fe++ in drainage fluid was generally higher than in plasma but was variable, (depending on the hemorrhage volume) and decreased over time.
Extracellular fluid volume, which makes up approximately 20% of body weight, is composed of 5% plasma (or intravascular fluid) and 15% interstitial fluid. The concentrations of Na+, K+, and Cl- in interstitial fluid are similar to those in plasma, while the total protein concentration of interstitial fluid is less than a third of that of plasma (Wait et al., 2001). It is therefore likely that our drainage fluids consisted primarily of interstitial fluid, with plasma composing the remaining ~20-40% of the total volume. However, drainage fluids are not simply a mixture of plasma and interstitial fluids because unlike these fluids, they also contain several other types of proteins, including various cytokine growth factors and chemokines.
Cytokine growth factor profiles in drainage fluids
The sequential changes in cytokine profiles in drainage fluids shown in this study clearly reflected the successive activities of various cells and the sequential phenomena involved in the wound healing process. Fig. 8 summarizes the sequential changes in the profiles of cytokines, chemokines, and MMPs, as measured in this study. In the early phase (postoperative days 0-1) of wound healing, b-FGF, PDGF, EGF, and TGF-β1 were present at high concentrations, and levels subsequently decreased acutely in the next stage of wound healing. Since b-FGF is known to be primarily derived from injured tissue or from cells infiltrating into wounds at early stages, tissue-bound b-FGF may be released after injury by several mechanisms, including cell lysis and cell injury (Muthukrishnan et al., 1991; Baker et al., 2000; Takamiya et al., 2002). PDGF, EGF, and TGF-β1 were detected at higher amounts in SPRP than in DF-E, suggesting that these growth factors were mainly supplied by dying, lytic platelets in the early phase of wound healing.
In the second phase of wound healing (postoperative days 2-4), KFG concentrations peaked, and those VEGF and HGF slightly increased. Later, in the third phase, (days 5-6) VEGF and HGF concentrations gradually increased to peak levels. Since these growth factors were present at only very low levels on days 0-1, it is possible that these increases resulted from their release from the cells that migrated to the wound site after day 1. KFG, VEGF, and HGF are thought to have roles primarily in granulation, angiogenesis and epithelialization (Peters et al., 1993; Gale et al., 1999; Lauer et al., 2000). KGF is known to be released mainly from fibroblasts and T cells (Brauchle et al., 1994; Marchese et al. 1995), VEGF from keratinocytes and macrophages (Brown et al., 1992; Frank et al. 1995), and HGF from mesenchymal cells such as dermal fibroblasts (Matsumoto et al., 1991).
Punctured fluids from subcutaneous seroma contained higher concentrations of growth factors seen in later phases such as VEGF, HGF, and KGF, but TGF-β1, which is seen in the early phase, was also abundant in seroma fluid. This finding may be based on different sources of the early-phase growth factors: PDGF and EGF are mostly derived from platelet, whereas TGF-β1 is supplied not only from platelets but also from various sources. The initial production of active TGf-β1 from platelets serves as a chemoattractant for neutrophils, macrophages, and fibroblasts, and these cells further enhance TGF-β1 production (Werner et al., 2002). As well as active forms, lent TGF-βs are also produced and sequestered within the wound matrix, allowing its sustained release by proteolytic enzymes (Gailit et al., 1994; Zambruno et al., 1995), and the combination of different cellular sources and temporary storage ensures a continuous supply of TGF-β1 throughout the repair process (Werner et al., 2002). A study using knock-out mice of TGF-β1 (Brown et al., 1995) showed that different growth factors can compensate for the lack of TGF-β1 in early wounds, but that TGF-β1 plays a crucial role later in the repair process.
Chemokine and MMP profiles in drainage fluids
We also examined IL-6 and IL-8, which are two pro-inflammatory chemokines (chemotactic cytokines). IL-6 is a major mediator of the host response to tissue injury (Sehgal, 1990), and is released by a variety of cell types including polymorphonuclear leukocytes, macrophages, fibroblasts, lymphocytes and endothelial cells (Panquet et al., 1996). IL-8 has been shown to stimulate chemotaxis of neutrophils (Hoch et al., 1996) and keratinocytes (Kemeny et al. 1994) and also to stimulate neovascularization (Koch et al., 1992). In our study, IL-6 was present in DF-E at about 7000 pg/mL and gradually decreased afterwards. Cells that appeared in the wound area in each phase seemed to be major sources of IL-6; neutrophils were present in the early phase and macrophages and lymphocytes were present in later phases (Fig. 8). In the case of IL-8, the concentration gradually increased up to day 6, so it is probable that the fibroblasts present in the second wave of cell migration to the wound produced significant amounts of IL-8, as was previously suggested in a study of fetal wound healing (Liechty et al., 1998).
Pro-inflammatory cytokines/chemokines directly stimulate the synthesis of the collagen-degrading matrix metalloproteinases (MMPs) and also inhibit the synthesis of tissue inhibitors of metalloproteinase in fibroblasts and endothelial cells (Murphy et al., 1994). We measured three types of collagenases, MMP-1, MMP-8 and MMP-13. MMP-1 preferentially degrades type III collagen (Welgus et al., 1981), whereas MMP-8 has its greatest activity on type I collagen (Hasty et al., 1987). MMP-13 can degrade all fibrillar collagen subtypes with nearly equal efficacy and is the only collagenase with significant activity against type II and IV collagen (Freije et al., 1994). Fibroblasts appear to be the cellular source for the majority of MMP-1, while neutrophils seem to provide most of MMP-8. In subcutaneous drainage fluids, MMP-1 gradually increased up to day 6, while MMP-8 peaked on days 2 to 3. At all time points examined, levels of MMP-8 were statistically significantly higher than both MMP-1 (50-fold to 200-fold) and MMP-13 (1000-fold to 10000 fold). The sequential changes that we observed in MMP-1 and MMP-8 were similar to previously reported data from a study of cutaneous wound fluids (Nwomeh et al., 1998). Taken together, these data suggest that MMP-8 functions as the primary debriding collagenase during the acute phase of wound healing.
Potential use of wound fluids and SPRP in culture media
Since our data showed that drainage fluids contained various growth factors which were not found in SPPP or SPRP, we tested drainage fluids as a substitute or supplement for serum in the culturing of ASCs and dermal fibroblasts. The experiment using ASCs showed that DF-E is superior to FBS as a 5% additive of the medium containing 5% FBS, while that using dermal fibroblasts suggested that drainage fluids may be used as similarly to serum. Thus, we suggest that drainage fluids may be used as a supplement or substitute for serum in culture media, and may be able to support the growth of cell types other than the two lines examined in this study.
Recent developments in the clinical use of cultured cells (such as stem cell therapy or gene delivery) have necessitated safer preparation and manipulation of cells, which partly entails avoiding the use of animal-derived serum, tissues and extracts. In this respect, autologous serum, cytokines or other soluble factors could be extremely valuable. For example, ASCs isolated from liposuction aspirates of a patient could be cultured using the patient’s own SPRP taken from blood and/or using drainage fluids taken from the subcutaneous wound after liposuction. Subcutaneous wound fluids have some advantages compared to cutaneous and intraperitoneal wound fluids: subcutaneous wound fluids are difficult to collected aseptically and in a large volume, and intraperitoneal ones can be obtained only through major abdominal surgery and are not aseptic in most cases.
Our results have shown that SPRP and drainage fluids can be abundant sources of autologous soluble factors such as cytokines, and our biochemical breakdown of DF-E, DF-L, SPPP, and SPRP provides the ability to assess the culture requirements of a particular cell line and then to select an appropriate fluid for use as a supplement or substitute for FBS in cell culture. Both SPRP and drainage fluids are economical ready-made mixtures of serum (plasma) and soluble factors such as cytokines, and can also be used as raw materials for the extraction of individual soluble factor proteins. Further investigation will be necessary to provide optimized protocols for the usage of drainage fluids and SPRP in cell culture and in factor isolation.

Figure Legends

Fig. 1
Biochemical profiles of preoperative serum and of drainage fluids on days 0 to 6.
Preoperative serum and drainage fluids were collected from the same three patients; drainage fluids were collected on days 0-6, where day 0 represents the day that surgery was performed. The preoperative serum value is indicated to the left on the x-axis and is labeled “serum”; the numbers 0-6 to the right represent day 0 through day 6 timepoints of drainage fluid collection. Values represent means ± S.E.

Fig. 2
Daily changes in cytokine concentrations in drainage fluids.
Drainage fluids were collected on days 0 to 6 from patients in the normal healing (NH) group, and cytokine concentrations were examined by ELISA. For VEGF and KGF, data was derived from six patients; for all other cytokines, data was derived from seven patients Each line shows data from one patient (e.g. 38F means 38 year-old female).

Fig. 3
Comparison of cytokine concentrations in DF-E, DF-L, PF, SPPP, and SPRP.
Cytokine concentrations were measured in drainage fluid samples from day 0 or day 1 (DF-E; Drainage Fluid-Early), drainage fluid from day 5 or day 6 (DF-L; Drainage Fluid-Late), punctured seroma fluid samples from days 14+ (PF; Punctured Fluids), serum from platelet-poor plasma (SPPP), and serum from platelet-rich plasma (SPRP). Cytokine concentrations in DF-E, DF-L, and PF were compared to those of SPPP and SPRP. Values represent means ± S.E. * P<0.05 (blue lines), ** P<0.01 (red lines).

Fig. 4
Changes in IL-6 and IL-8 concentrations in drainage fluids and comparison to PF, SPPP and SPRP.
The concentration of IL-6 and IL-8 was measured by ELISA in drainage fluids collected on days 0-6 and was similarly measured in PF, SPPP, and SPRP. Data of daily changes from seven patients were shown in left figures, in which each line shows data from one patient (e.g. 38F means 38 year-old female). Values in right graphs represent means ± S.E. * P<0.05 (blue lines), ** P<0.01 (red lines).

Fig. 5
Changes in concentrations of MMP-1, MMP-8, and MMP-13 in drainage fluids and comparison to PF, SPPP and SPRP.
The concentration of MMP-1, MMP-8, and MMP-13 was measured by ELISA in drainage fluid collected on days 0-6 and in seroma puncture fluid (PF), SPPP, and SPRP. Data of daily changes from seven patients were shown in left figures, in which each line shows data from one patient (e.g. 38F means 38 year-old female). Values in right graphs represent means ± S.E. * P<0.05 (blue lines).

Fig. 6
Proliferation of ASCs and dermal fibroblasts.
ASCs (A) and dermal fibroblasts (B) were cultured with DMEM containing various amounts of FBS and/or drainage fluids (DF-E or DF-L) for 1 week and cell numbers were counted. Each cell number was expressed as a ratio to that of the control culture, which was grown in media that contained 5% FBS and lacked drainage fluid. Values represent means ± S.E. * P<0.05 (bracketed blue lines), ** P<0.01 (bracketed red lines).

Fig. 7
Comparison of cytokine concentrations with data of previous studies.
Data from this study on cytokine concentrations in drainage fluids from subcutaneous wounds were compared with data from two previous studies examining drainage fluids from intraperitoneal wounds (Baker et al., 2003) and fluids from cutaneous wounds; the donor sites of split skin grafts (Vogt et al., 1998). Values represent means ± S.D.

Fig. 8
Summary of sequential changes in soluble factors associated with wound healing in drainage fluids from subcutaneous wounds.
There are three types of sequential changes in the abundance of soluble factors that function in wound healing. First, levels of b-FGF, EGF, PDGF and TGF-β are initially high and then gradually decrease. EGF and PDGF in drainage fluids in the early phase (coagulation phase) of wound healing are derived from platelets, although TGF-β is derived from various sources, and b-FGF is mainly derived from injured tissue or from cells infiltrating into wounds at early stages. Second, KGF, IL-6, and MMP-8 peak around days 2 to 4 (during the inflammatory phase). KGF is released from T lymphocytes and fibroblasts, while IL-6 seems to be discharged from the various cells involved in each phase. Third, VEGF, HGF, IL-8, and MMP-1 are low in the early phase and gradually increase up to the late phase (proliferation phase). These factors are derived from cells involved in the later phases of wound healing, including fibroblasts and keratinocytes. IGF-1 is present at relatively consistent levels throughout the entire wound healing process. MMP-13 is detected only in minimal quantities.

Table 1
Sources of autologous soluble factors associated with wound healing: comparison of drainage fluids, SPPP and SPRP.
Relative abundances are indicated by -, +/-, +, and ++. ++ indicates high abundance of a factor, and ? indicates absence of a factor.


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